Plant Physiology Preview. Published on April 28, 2017, as DOI:10.1104/pp.16.01983
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Short title: Concerted divergence of duplicated genes
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Concerted divergence after gene duplication in Polycomb Repressor
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complexes
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Yichun Qiu , Shao-Lun Liu , and Keith L. Adams
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1Department of Botany, University of British Columbia, Vancouver, British Columbia, Canada
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2Department of Life Science, Tunghai University, Taichung, Taiwan
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Corresponding author: Keith L. Adams, E-mail: [email protected]
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Summary: FIS2 and MEA have diverged in concert after simultaneous gene duplication,
resulting in functional divergence of the PRC2-complexes in Brassicaceae, which is a novel fate
for duplicated genes whose products act in complexes.
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Author contributions: Y.Q. and K.L.A. designed the research. Y.Q. and S.-L.L. performed the
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experiments and analyzed the data. Y.Q. and K.L.A. wrote the manuscript.
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Funding information: This work was supported by a Discovery Grant from the Natural Science
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and Engineering Research Council of Canada (to KLA), a Postgraduate Fellowship from NSERC
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(to YQ), and grants from the Ministry of Science and Technology, Taiwan (to SLL).
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Abstract
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Duplicated genes are a major contributor to genome evolution and phenotypic novelty. There are
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multiple possible evolutionary fates of duplicated genes. Here we provide an example of
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concerted divergence of simultaneously duplicated genes whose products function in the same
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complex. We studied PRC2 (Polycomb Repressive Complex 2) in Brassicaceae. The VRN-PRC2
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complex contains VRN2 and SWN, and both genes were duplicated during a whole genome
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duplication to generate FIS2 and MEA which function in the Brassicaceae-specific FIS-PRC2
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complex that regulates seed development. We examined expression of FIS2, MEA, and their
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paralogs, compared their cytosine and histone methylation patterns, and analyzed the sequence
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evolution of the genes. We found that FIS2 and MEA have reproductive-specific expression
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patterns that are correlated and derived from the broadly expressed VRN2 and SWN in outgroup
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species. In vegetative tissues of Arabidopsis repressive methylation marks are enriched in FIS2
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and MEA, whereas active marks are associated with their paralogs. We detected comparable
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accelerated amino acid substitution rates in FIS2 and MEA but not in their paralogs. We also
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show divergence patterns of the PRC2-asssociated VEL2 that are similar to FIS2 and MEA.
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These lines of evidence indicate that FIS2 and MEA have diverged in concert, resulting in
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functional divergence of the PRC2-complexes in Brassicaceae. This type of concerted
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divergence is a previously unreported fate of duplicated genes. In addition, the Brassicaceae-
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specific FIS-PRC2 complex modified the regulatory pathways in female gametophyte and seed
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development.
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Introduction
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Duplicated genes are continuously formed during evolution by various types of gene duplication
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events in eukaryotes and they can have effects on morphological and physiological evolution
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(reviewed in Van de Peer et al., 2009; Soltis and Soltis 2016). Gene duplication can happen at
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small scales, such as tandem duplication, segmental duplication, and duplicative retroposition.
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The largest scale of gene duplication is whole genome duplication (WGD), which gives rise to
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thousands of duplicated gene pairs. The genetic model plant, Arabidopsis thaliana, has
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experienced five rounds of WGD events in the evolutionary history of seed plants (Jiao et al.,
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2011; Li et al., 2015). The most recent polyploidy event, the alpha WGD, is specific to the
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Brassicaceae family, which took place after the divergence of the closest sister family,
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Cleomaceae (Schranz and Mitchell-Olds, 2006). There are about 2500 pairs of duplicated genes
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retained from this WGD in the Arabidopsis thaliana genome (Blanc et al., 2003; Bowers et al.,
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2003).
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Fates of duplicated genes vary during evolutionary history. One duplicate may eventually
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be lost or become a pseudogene, thus the once duplicated pair returns to a single-copy status.
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Several mechanisms drive the retention of both copies. Duplicated pairs could preserve similar
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functions to maintain dosage balance (Birchler et al., 2005; Coate et al., 2016). Duplicated pairs
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can also diverge through subfunctionalization or neofunctionalization, where two duplicated
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genes divide the ancestral function or gain a novel function, respectively (Force et al., 1999;
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Moore and Purugganan, 2005). These types of divergence could also be inferred from expression
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pattern. For example, two duplicates together make up the pre-duplicate expression profile is
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referred to as regulatory subfunctionalization, and regulatory neofunctionalization indicates one
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or both copies gain a new expression pattern (Duarte et al., 2006; Liu et al., 2011). Sometimes
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these processes are difficult to distinguish, and there can be a combination of different
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mechanisms such as sub-neofunctionalization (He and Zhang, 2005).
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There are many protein complexes whose members are encoded by different gene
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families. If multiple components in a complex are duplicated simultaneously, such as in a whole
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genome duplication, the doubled components could redundantly cross-interact, or go on to
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experience subsequent divergence (Capra et al., 2012; Aarke et al., 2015). Thus a type of co-
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evolution between the interacting gene products is hypothetically possible, but has not been
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described in the plant kingdom. Extending the concept of concerted divergence, which is
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discussed in the context of co-expression patterns of duplicated genes in the same metabolic or
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regulatory pathways (Blanc and Wolfe, 2004), we here propose the evolutionary scenario that
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simultaneous duplication of two genes whose products function together in a complex, followed
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by parallel evolution and divergence of each derived gene, can lead to functional divergence of
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the complexes.
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In this study we focus on genes in PRC2 complexes (Polycomb Repressive Complex 2)
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in Brassicaceae species as a potential example to demonstrate the proposed scenario. Those
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complexes are histone modifiers, and regulate gene expression primarily by tri-methylation of
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lysine 27 on histone H3 (H3K27me3) associated with target genes which leads to transcriptional
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repression (Hennig and Derkacheva, 2009; Mozgova et al., 2015). One type of PRC2, the VRN-
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complex that is present across all rosids, regulates vegetative tissue differentiation, and more
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importantly, vernalization process to control flowering time in Arabidopsis (Chen et al., 2009;
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Hennig and Derkacheva, 2009; Mozgova et al., 2015). The complex also represses autonomous
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seed coat development (Roszak and Kohler, 2011) and it is present across rosids. The VRN2
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complex consists of four subunits: REDUCED VERNALIZATION RESPONSE 2
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(VERNALIZATION2, VRN2), SET DOMAIN-CONTAINING PROTEIN 10 (SWINGER,
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SWN), with two WD-40 repeat proteins who act as the scaffold of the complex assemblies,
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FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) and MULTICOPY SUPRESSOR OF
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IRA1 (MSI1). In Brassicaceae, the alpha WGD gave rise to a duplication of VRN2 to create its
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paralog FERTILIZATION INDEPENDENT SEED 2 (FIS2) and a duplication of SWN to create its
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paralog SET DOMAIN-CONTAINING PROTEIN 5 (MEDEA, MEA) (Fig. 1; Luo et al., 2009;
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Spillane et al., 2007). Substituting for their paralogous proteins, FIS2 and MEA, together with
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FIE and MSI1 (the alpha WGD paralogs of these two genes were lost), make up a new
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Brassicaceae-specific PRC2, referred to as the FIS-complex (Fig. 1). The FIS-complex functions
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in gametophyte and seed development, preventing female gamete proliferation before
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fertilization, and facilitating endosperm cellularization after fertilization (Hennig and
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Derkacheva, 2009). A typical fis phenotype, caused by non-functional mutation in FIS2, MEA
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(also known as FIS1) or FIE (also known as FIS3), shows fertilization independent
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embryogenesis, and other types of mutants have abnormal seed development, even abolished
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seeds (Hennig and Derkacheva, 2009).
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The observed divergence in the functions of the two kinds of PRC2 complexes leads to
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the hypothesis that FIS2 and MEA have undergone divergence in a concerted way to give rise to
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the FIS-complex. This study aimed to evaluate this hypothesis by examining expression patterns,
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DNA and histone methylation, and rates of sequence evolution in both genes compared with
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their paralogs. We found evidence for parallel divergence of FIS2 and MEA from their paralogs
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in multiple ways that has accompanied functional divergence of the two complexes. This study
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supports a model of concerted divergence of simultaneously duplicated genes whose products
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function in a complex. This is a previously unreported fate of duplicated genes.
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Results
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FIS2 and MEA have specific and similar expression patterns in reproductive organs
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FIS2 and MEA formed by the alpha whole genome duplication that is specific to the
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Brassicaceae family after the divergence of the Brassicaceae lineage from the Caricaceae lineage.
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After gene duplication, duplicated genes may experience expression divergence. We analyzed
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microarray data in Arabidopsis thaliana to compare the expression profiles of paralogous
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interacting gene pairs FIS2/VRN2 and MEA/SWN. We obtained two sets of ATH1 microarray
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data and analyzed them separately: 63 different organ types and developmental stages (Schmid et
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al., 2005), referred to as ADA (Arabidopsis developmental atlas) dataset hereafter, and 42
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different tissue types during seed developmental stages (Le et al., 2010), referred to as ASA
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(Arabidopsis seed atlas). We first calculated the expression specificity (τ) of the four genes
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defined by Yang and Gaut (2011). VRN2 and SWN have expression specificity values of 0.19 and
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0.17 respectively, indicating that both genes have relatively broad expression in nearly all organ
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types included in the ADA dataset. In contrast, FIS2 has an expression specificity value of 0.70
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and MEA is 0.63, indicating an organ-specific expression pattern. We observed that the
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expression of FIS2 and MEA is restricted to flowers and siliques, and the absence of vegetative
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expression explains the high expression specificity. Yang and Gaut (2011) analyzed the ADA
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dataset and they found that the recent WG duplicates have a median tau close to 0.2. Thus what
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we observed for FIS2 and MEA is quite high, and what we observed for VRN2 and SWN is about
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average.
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We also analyzed expression specificity (τ) in the ASA dataset (Fig. 2A). Similarly, the τ of FIS2
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is 0.48 and MEA is 0.56, while VRN2 has τ of 0.21 and SWN has 0.22. FIS2 and MEA turn out to
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show more tissue-specific expression in seed tissues. We broke down the ASA data and observed
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that FIS2 and MEA tend to be expressed in the triploid endosperm rather than in the diploid
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embryo or maternally derived seed coat. We did a 1000-replicate permutation test and gained
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statistical support that the expression specificity differences in the FIS2-MEA and VRN2-SWN
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comparisons are not significant (Fig. S1), indicative of the concerted divergence in their
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expression profile. In contrast, the tissue specificity expression profile is significantly different in
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the two duplicated pairs, VRN2-FIS2 and SWN-MEA, indicative of their regulatory divergence.
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Not only did we analyze the expression index for those genes individually, we also
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performed a correlation test to examine the association of the expression profiles of the four
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genes, as their products function in a complex (Fig. 2B). We found that the expression patterns
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of FIS2 and MEA are positively correlated in both the ADA and ASA datasets, while broadly
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expressed VRN2 and SWN are co-expressed. However, the expression of both FIS2 and MEA is
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negatively correlated to the expression of VRN2 and SWN. The negative coefficients are around
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-0.5 (Fig. 2B), which is below 1% of the total alpha WG pairs analysed by Blanc and Wolfe
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(2004). Overall the FIS2-MEA expression patterns indicate parallel divergence from VRN2-SWN
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expression patterns in a concerted manner.
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FIS2 and MEA acquired new expression patterns
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As the microarray data from the Arabidopsis thaliana developmental expression atlas indicated,
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FIS2 and MEA both have an expression pattern that is restricted to reproductive organs, such as
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flowers and siliques, but not vegetative organs, including roots, stems, and leaves. We confirmed
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this result with RT-PCR (Fig. 3). In contrast, their paralogs, VRN2 and SWN, have a broad
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expression pattern in both vegetative and reproductive organs, and they are ubiquitously
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expressed in all examined organ types in our RT-PCR results (Fig. 3). To infer the ancestral
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expression pattern of the two gene pairs, we assayed the expression pattern of orthologs in
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Tarenaya hassleriana (formerly known as Cleome spinosa), Carica papaya and Vitis vinifera.
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Among those species with sequenced genomes, Tarenaya belongs to Cleomaceae, the most
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closely related sister group to Brassicaceae. Although Tarenaya has its own genome triplication
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after the divergence between Cleomaceae and Brassicaceae (Cheng et al., 2013), only a single
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copy each of the orthologous VRN2 and SWN has been retained. Carica is also in the order
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Brassicales. Vitis was chosen because its lineage has not experienced any whole genome
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duplication events since the gamma WGD during early eudicot evolution, which applies to
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Carica as well, and thus genes are frequently single copy in these taxa. These single-copy
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orthologs can facilitate the inference of ancestral expression pattern. We confirmed that these
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sequences are true orthologs of FIS2/VRN2 and MEA/SWN by phylogenetic analysis of the gene
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families.
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For both the FIS2/VRN2 and MEA/SWN pairs, their orthologs in Tarenaya, Carica and
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Vitis are widely expressed in all examined organ types, which is the same as VRN2 and SWN in
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Arabidopsis (Fig. 3). The absence of expression in vegetative organs is only observed in FIS2
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and MEA. Collectively we inferred that the pre-duplicated expression state is likely to be a broad
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expression pattern, which is reflected by VRN2 and SWN. The Brassicaceae FIS2 and MEA both
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lost expression in vegetative organs to become specifically expressed in reproductive organs.
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FIS2 and MEA acquired novel epigenetic modifications
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The epigenetic features of cytosine methylation and histone methylation are often associated
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with expression or silencing of genes. To examine the patterns of cytosine and histone
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methylation in organ types where expression of FIS2 and MEA was lost, we investigated the
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epigenetic variation among these genes in vegetative tissues including leaves, roots and seedlings
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of Arabidopsis thaliana (see methods for details). For DNA methylation, we found that cytosine
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methylation at CpG sites is enriched in the promoter region (defined as 1500 bp upstream of the
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transcription start site) of FIS2 genomic sequence, but not the gene body (Fig. 4). The opposite is
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found for VRN2, with the promoter region unmarked but the gene body is highly methylated (Fig.
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4). The same divergence of DNA methylation was found for MEA and SWN (Fig. 4). Cytosine
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methylation is enriched in the promoter region of MEA but only in the gene body of SWN. The
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DNA methylation patterns in EMF2 and CLF, the more distant paralogs of VRN2 and SWN,
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respectively, are also gene body enrichment, the same as VRN2 and SWN, suggesting that the
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pattern of DNA methylation for FIS2 and MEA has changed after duplication. As promoter
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cytosine methylation is associated with transcriptional repression, and gene body methylation is
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indicative of expression activation (Suzuki and Bird 2008), this finding is consistent with the
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expression data. We did not examine methylation patterns in whole endosperm because in the
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ASA seed atlas dataset FIS2 and MEA showed variable expression patterns in different parts of
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the endosperm and different developmental stages.
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We also examined histone methylation in the region of these genes in the seedlings of
Arabidopsis thaliana based on the data generated by Roudier et al. (2011). Similar to DNA
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methylation, we found that VRN2, SWN, EMF2 and CLF have the same types of histone
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methylation, which are different from FIS2 and MEA (Table 1). We noticed that FIS2 and MEA
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lost H3K4me3, which is shared by all the other genes. Instead they gained a novel mark of
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H3K27me3. H3K4me3 is an activating mark, while its antagonistic mark H3K27me3 is
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repressive. This could help explain the expression of VRN2, SWN, EMF2 and CLF in the
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vegetative tissue, but the lack of expression of FIS2 and MEA. It is also notable that in the fie
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mutant, where the PRC2 function was supposed to be abolished, FIS2 and MEA lost their
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H3K27me3, but instead VRN2, SWN, EMF2 and CLF were marked by H3K27me3 (Bouyer et al.,
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2011). As H3K27me3 is regulated by PRC2 complexes, this finding suggests the self- and cross-
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regulation among these genes. With both DNA and histone modification comparative analyses
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we observed the convergent evolution of epigenetic features in FIS2 and MEA, divergent from
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their pre- and post-duplicated paralogs.
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Gene structural changes in FIS2 and MEA
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FIS2 formed from VRN2 by duplication, and MEA duplicated from SWN, during the alpha whole
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genome duplication. FIS2 in Arabidopsis thaliana lost three exons, called the E15-17 region
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(exons 15 to 17) (corresponding to the 15th to 17th exons in the Arabidopsis thaliana EMF2, not
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named after VRN2) compared to VRN2 (Fig. S2A; Chen et al., 2009). FIS2 has a large serine-rich
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domain that is not shared with any other VEF genes in any species, indicating gain of the domain
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in Brassicaceae (Fig. S2A; Chen et al., 2009). Our sequence analysis showed that the serine-rich
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domain is highly variable among FIS2 sequences from different Brassicaceae species (Fig. S2A).
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The lost E15-17 domain and the gained serine-rich domain are both neighbouring the VEF
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domain that interacts with the C5 domain in MEA.
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MEA is about 150 aa shorter than SWN, and the deleted region is just downstream of the
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C5 domain which interacts with the VEF domain in FIS2, due to a large shrinkage in a single
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exon (the 9th in Arabidopsis thaliana MEA and SWN) where Brassicaceae SWN and
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orthologous SWN-like sequences are not conserved (Fig. S2B). How the structural changes
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affect the physical interaction of FIS2 and MEA remains to be tested. In addition to the
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rearrangement of functional domains, those shared domains show different levels of amino acid
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sequence divergence. In contrast, VRN2/EMF2-like sequences and SWN/CLF-like sequences
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show relative conservation across all flowering plants in amino acid sequences and functional
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domains (Chen et al., 2009; Qian et al., 2014).
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FIS2 and MEA show accelerated amino acid substitution rates and evidence for positive
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selection
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Duplicated genes not only diverge in expression pattern but also in their sequences. We first
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analyzed by Ka/Ks analysis the full-length coding region of FIS2, VRN2, MEA, and SWN genes
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(Fig. S3). The Brassicaceae FIS2 clade had a much higher average Ka/Ks than VRN2 lineages,
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3.5-fold greater than the paralogous Brassicaceae VRN2 clade, and 10-fold greater than the
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orthologous pre-duplicate VRN2 sequences. Similarly, the Brassicaceae MEA clade had a high
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average Ka/Ks comparable to the FIS2 clade, which is 3.5-fold greater than the paralogous
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Brassicaceae SWN clade, and 4.5-fold greater than the orthologous pre-duplicated SWN
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sequences. We implemented different models assuming similar vs. different Ka/Ks ratios in these
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clades, described in the methods section, and the likelihood ratio tests indicated that the
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divergence in sequence rate is significant (Table S1). These analyses indicate that while the
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paralogous Brassicaceae VRN2 and SWN lineages are under stronger purifying selection along
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with the orthologous genes in outgroup species, FIS2 and MEA in the Brassicaceae have
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experienced relaxation of purifying selection. Asymmetric Ka/Ks ratios are seen in a minority of
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duplicated gene pairs in Arabidopsis thaliana; for example, Gossmann and Schmid (2011)
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estimated that 7% of the duplicated pairs they analyzed have asymmetric Ka/Ks ratios.
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Additionally, among the branch-wise Ka/Ks of specific FIS2 and MEA sequences, we
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detected possible positive selection, indicated by Ka/Ks greater than one, acting on the sequences
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from certain lineages (Fig. S3). In order to distinguish certain amino acid sites evolving under
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positive selection from relaxed purifying selection, we also applied a branch-site model, which
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suggested that both branches leading to Arabidopsis FIS2 (P<0.0001) and MEA (P=0.007) have
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positively selected amino acid sites across different functional domains (Fig. S4).
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Thus we further studied the sequence evolution of characterized functional domains of
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FIS2/VRN2 and MEA/SWN genes, including the VEF and C2H2 domains in the FIS2 and VRN2
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genes, and the C5, SET, SANT and CXC domains in the MEA and SWN genes (Fig. 5, Fig. S3).
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We observed that the trend of acceleration in sequence evolution of FIS2 and MEA, and
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evolutionary constraint resulting in the conservation of VRN2 and SWN, was reflected by all the
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functional domains we analyzed individually. The VEF domain in FIS2/VRN2 genes and the C5
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domain in MEA/SWN genes physically interact with each other, thus the comparison between the
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two sets of Ka/Ks ratios best describes the co-evolution between FIS2 and MEA at the coding
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sequence level from a protein-protein interaction perspective (Fig. 5). Consistent with the full-
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length gene analyses, the VEF domain in the FIS2 lineages and the C5 domain in the MEA
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lineages both have accelerated amino acid substitution rates, with evidence (Ka/Ks > 1)
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suggesting positive selection on a few branches (Fig. S3; Table S1). Similar results were found
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in the DNA binding related domains, C2H2 in FIS2/VRN2, CXC and SANT in MEA/SWN genes
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(Fig. S3), indicating that the PRC2 complexes with FIS2 and MEA may have affinity to specific
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DNA regions, regulating a novel network of gene expression. The SET domain plays the role of
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methyltransferase in the PRC2 complex, and is usually highly conserved across eukaryotes
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(Baumbusch et al., 2001). This is reflected by the low Ka/Ks ratios detected in the SWN SET
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domains (Fig. S3). Instead, the SET domain in the Brassicaceae MEA shows evidence for
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positive selection (Fig. S4). The rapid amino acid substitution rates in the PRC2 functional
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domains together likely relate to the functional divergence of the PRC2 complexes containing
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FIS2 and MEA.
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VEL2 and VEL1, which interact with PRC2 complexes, show corresponding divergence
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patterns to FIS2/VRN2 and MEA/SWN
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A family of five PHD finger proteins is necessary for the core PRC2 complex to maintain the
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repressed status of chromatin (Kim and Sung, 2010; Kim and Sung, 2013). Among them,
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VERNALIZATION5/VIN3-LIKE 1 (VEL1) and VEL2 are a pair of alpha whole genome duplicates.
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VEL2 is a maternally expressed imprinted gene (Wolff et al. 2011). We analyzed their expression
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profile in the ADA and ASA microarray datasets, and detected that VEL1 shows a co-expression
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pattern with VRN2 and SWN, which is similar to the broadly expressed VEL homologs, whereas
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VEL2 has a similar expression pattern to FIS2 and MEA due to loss of vegetative expression (Fig.
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6A; Qiu et al., 2014). VEL2 has a higher specificity than its paralog VEL1 (Fig. 6B). Thus the
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observed concerted divergence in expression pattern in the FIS-complex is not limited to the core
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complex, but also includes other associated proteins.
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For cytosine methylation in the vegetative tissue, VEL1 is marked through the coding
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exons but not the promoter region, whereas VEL2 has cytosine methylation enriched in the
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upstream promoter region and the first two introns located between the 5’UTR exons (Schmitz et
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al., 2013; Stroud et al., 2013; Zemach et al., 2013). For histone methylation in the vegetative
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tissue, VEL1 is marked by activating marks, including H3K4me3, H3K36me3 and H3K4me2
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(Roudier et al., 2011). VEL2 has lost the H3K4me3 and H3K36me3, but instead gained the
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repressive mark H3K27me3. Those epigenetic features not only correspond to the vegetative
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expression level, but also are consistent with the divergence of the core PRC2 components FIS2
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and MEA (Table 1).
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We further analyzed the sequence evolution of the VEL genes. The VEL2 sequences have
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an elevated average Ka/Ks ratio compared to the Brassicaceae VEL1 and orthologous VEL genes
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(Fig. 6B). While VEL1 and orthologous sequences have a low Ka/Ks ratio close to zero
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indicating strong purifying selection, a three-fold change in VEL2 sequences suggests the
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relaxation of purifying selection. This coincides with the accelerated amino acid substitution
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rates of FIS2 and MEA.
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Discussion
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Concerted divergence of FIS2 and MEA in the FIS-PRC2 Complex
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Upon gene duplication, hypothetically two duplicates are identical in function, as well as
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expression pattern if the cis-elements also are entirely duplicated. Considering that many
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proteins function through interactions with other proteins, in a regulatory or metabolic pathway,
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through protein-protein interaction, or form an integral complex, either the duplicates are
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redundant or both duplicates could integrate into either complex and affect the function of the
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complex if they have divergence. A shift in expression pattern would be one way to avoid
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potentially disadvantageous crosstalk between interacting members (Aarke et al., 2015). Blanc
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and Wolfe (2004) described a process of concerted divergence of gene expression in Arabidopsis
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thaliana, in which pairs of duplicates, whose protein products interact, diverge in a parallel
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manner in expression pattern. However, as FIS2 and VRN2 were not identified as alpha WG
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duplicates by the genome-wide study (Blanc et al., 2003), their concerted divergence in
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expression pattern with MEA and SWN was not included.
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Here we show that FIS2 and MEA diverged in expression pattern in a concerted manner,
329
modified from co-expressed VRN2 and SWN whose expression pattern resembles the ancestral
330
status. In addition, we show that cytosine methylation and histone methylation patterns in FIS2
331
and MEA also diverged in a concerted manner. It is possible that the methylation change
332
contributed to the changes in expression patterns, although mutations in regulatory elements may
333
also have played a role in the expression pattern changes. FIS2 and MEA are marked by
334
H3K27me3 in the vegetative tissue, suggesting they both became the targets of a vegetative
335
PRC2 complex after formation by gene duplication (Bouyer et al., 2011). In addition to the
336
vegetative epigenetic divergence, FIS2 and MEA are well known as imprinted genes during seed
337
development, both of which are maternally expressed genes (Berger and Chaudhury, 2009).
338
Based on the genome-wide datasets from Hsieh et al. (2011) and Gehring et al. (2011), we
339
determined that VRN2 and SWN are not imprinted, while the more distant relatives in their gene
340
families, EMF2 and CLF, also lack of evidence for imprinting. Thus we infer that FIS2 and MEA
341
became imprinted genes after their divergence from VRN2 and SWN. This concerted change in
342
regulation of both genes ensures the dosage balance between the interacting proteins. The
343
concerted divergence of FIS2 and MEA from their paralogs is also reflected by the elevated
16
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344
Ka/Ks ratios in the coding sequences at comparable levels, suggesting similar relaxed purifying
345
selection is acting on the two genes. Altogether these changes indicate that FIS2 and MEA have
346
been diverging in concert in multiple ways, which likely contributed to the divergence in
347
functions between the FIS2-PRC2-complex and the VRN2-PRC2-complex.
348
349
Functional divergence in the FIS-PRC2 Complex
350
VRN2, SWN/CLF, FIE, and MSI1 form the VRN-complex, which regulates vernalization to
351
control flowering time in Arabidopsis (Fig. 1; Hennig and Derkacheva, 2009). The complex also
352
represses autonomous seed coat development (Roszak and Kohler, 2011). The FIS-complex
353
contains FIS2, MEA, FIE, and MSI1. The FIS-complex is important in gametophyte and seed
354
development and it has two major functions. A pre-fertilization role for the FIS-complex is that it
355
prevents proliferation of the central cell of the female gametophyte until after fertilization so that
356
seed development does not start until after fertilization (Hennig and Derkacheva, 2009). The
357
FIS-complex also acts post-fertilization. It is needed for regulating endosperm cellularization
358
during seed development (Hehenberger et al., 2012). FIS2 mutants show a phenotype of
359
abnormal female gametophyte development into embryos and are defective in controlling central
360
cell proliferation in the female gametophyte, suggesting that FIS2 is not redundant with VRN2 in
361
the pre-fertilization function (Roszak and Kohler, 2011). Thus the FIS-complex function in the
362
female gametophyte is specific to the FIS-complex and not the VRN-complex. MEA was also
363
shown to not be redundant with SWN (Roszak and Kohler, 2011). Unlike all the key components
364
in FIS-complex, a SWN mutant failed to lead to autonomous seed development in the absence of
365
fertilization, nor seed abortion with embryo and endosperm overgrowth (Luo et al., 1999), thus it
366
is possible that MEA is functionally specialized for the pre-fertilization function of the FIS-
367
complex and can not be complemented by SWN. As for the post-fertilization function, SWN was
368
shown to be not essential in seed development (Spillane et al., 2007). Thus it was proposed that
369
MEA underwent neofunctionalization to gain a post-fertilization role in regulating seed
370
development after its duplication from SWN (Spillane et al., 2007). Taking the two parts of the
371
FIS-complex functions together, it appears that the novel PRC2 made up by FIS2 and MEA
372
created a Brassicaceae-specific complex for preventing seed development prior to fertilization
373
and facilitating seed development after fertilization in Brassicaceae. This functional divergence
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374
complements the concerted divergence of FIS2 and MEA in other ways that we show in this
375
study. The FIS-complex also plays an important role in establishing imprinted expression of
376
many genes in the endosperm, especially paternally expressed imprinted genes, as the
377
differentially methylated paternal or maternal allele can affect the targeting by this complex
378
(Wolff et al., 2011; Kohler et al., 2012). The concerted divergence of FIS2 and MEA in
379
expression patterns, methylation patterns, and accelerated sequence evolution may have
380
contributed to functional diversification or potentially neofunctionalization of the FIS-PRC2
381
complex. An alternative to neofunctionalization of the FIS-PRC2 complex is
382
subfunctionalization after the formation of FIS2 and MEA from their paralogs. Without
383
knowledge of the ancestral function of the PRC2 complex in plants closely related to the
384
Brassicaceae, discussed below, we cannot say for sure if there has been neofunctionalization or
385
subfunctionalization. We show in this study that there has been regulatory neofunctionalization
386
of FIS2 and MEA, which leads us to favor the possibility of neofunctionalization of the complex.
387
Nonetheless, under a scenario of subfunctionalization, FIS2 and MEA still show concerted
388
divergence in their expression patterns, cytosine and histone methylation, and accelerated
389
sequence evolution. In order to distinguish the two possible hypotheses, more research on VRN-
390
complexes in rosid species will provide valuable information to infer the function of the ancestral
391
rosid PRC2 complex.
392
How are the FIS-complex functions performed in other angiosperms outside of
393
Brassicaceae? Some clues come from studies of FIE, which is a member of the FIS2 complex, in
394
Hieracium piloselloides (Asteraceae). The central cell proliferation phenotype of Arabidopsis fie
395
mutants is not seen in sexual Hieracium FIE RNAi lines; thus a PRC2 complex does not regulate
396
central cell proliferation in the female gametophyte of Hieracium, in contrast to Arabidopsis
397
(Rodrigues et al., 2008). This might indicate that parts of the pre-fertilization function of FIS-
398
PRC2 in Brassicaceae is an evolutionary innovation, at the same time it is possible the unknown
399
mechanism repressing central cell proliferation is specific to the Hieracium lineage. FIE down-
400
regulation in Hieracium leads to seed abortion (Rodrigues et al., 2008) and thus FIE is important
401
for seed development, presumably as part of a PRC2 complex. Asterids do not contain FIS2,
402
VRN2, or MEA. Thus, if there is a PRC2 complex regulating seed development in asterids, it
403
probably contains the product of lineage specific polycomb proteins, and a mechanism
404
independently evolved from Brassicaceae. In maize and rice there has been duplication of FIE
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405
(Luo et al., 2009; Li et al., 2014). Thus the grasses may have PRC2 complexes that are
406
divergent from the ancestral state. The requirement of H3K27me3 in rice and maize endosperm
407
for establishment of imprinting suggests the functional conservation or convergence of a PRC2
408
complex in Brassicaceae and Poaceae (Makarevitch et al., 2013; Zhang et al., 2014).
409
410
Evolution of protein complexes after the duplication of components
411
We propose the model of simultaneous gene duplication and concerted divergence of one copy
412
of each duplicated pair (Fig. 7). Following formation by duplication, two genes whose products
413
function together in a complex diverge in similar ways and the complex diverges in function.
414
This divergence pattern is not limited to neo- / sub-functionalization, but includes some other
415
modifications of these scenarios such as escape from adaptive conflict. The PRC2 complexes in
416
Brassicaceae we examined in this study provide the first example of this type of divergence of
417
duplicated genes. We contrast this scenario with single-gene-duplication and divergence, where
418
one component in the complex underwent gene duplication, then the paralog diverges driving the
419
two complexes with either paralog to diverge in function as a result. Intuitively many described
420
functionally divergent paralogs may contribute to this type of divergence of their protein
421
complexes. One example is the centromere-defining histone variants CENH3 in the histone core
422
octamers that show duplication specific to the genus Mimulus and sequence divergence, whereas
423
other components in the histone core octamers do not show duplications specific to Mimulus
424
(Finseth et al., 2015). Another case is the telomere-associated proteins POT1a and POT1b in the
425
telomerase RNP complexes in Brassicaceae, where POT1a experienced positive selection that
426
enhanced its affinity with interacting proteins (Beilstein et al., 2015). A variation on this model
427
is when there is a subsequent gene duplication at a later time of another gene whose product
428
functions in the complex, followed by divergence. An example is the plant-specific RNA
429
polymerase IV and V where rounds of independent lineage-specific duplications and subsequent
430
divergence of varying kinds of subunits have increased RNA polymerase complexity and
431
specificity among different plant groups (Wang et al., 2015).
432
433
Concerted divergence of the functionally associated VELs and some PRC2 targets
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434
The VEL genes, VEL1 and VEL2, which are required to maintain and facilitate polycomb
435
transcriptional repression, interact with the PRC2 complex but are not part of the complex itself.
436
Our expression, methylation, and sequence analysis results indicate that VEL2 has similar
437
patterns to FIS2 and MEA, whereas VEL1 has similar patterns to VRN2 and SWN. Thus VEL2
438
appears to be diverging in concert with FIS2 and MEA. VEL2 is also a maternally expressed
439
gene and regulated by the FIS-complex in the endosperm (Wolff et al., 2011), and VEL2 works
440
together with the FIS core complex to impose maternal regulation in seed development similar to
441
FIS2 and MEA.
442
Several PRC2 targets duplicated through the alpha WGD show similar patterns of
443
divergence as well. PKR2 and JMJ15 are FIS-PRC2 regulated imprinted genes (Hsieh et al.,
444
2011; Wolff et al., 2011), whereas their paralogs, PKL and JMJ18 show broad expression, are
445
not imprinted, and are associated with a vegetative PRC2 complex (Aichinger et al., 2011; Yang
446
et al., 2012; Zhang et al., 2012). Out of 46 imprinted genes regulated by FIS2 (Wolff et al.,
447
2011), we identified 41 Brassicaceae-specific duplicated genes. Some of those genes have roles
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448
in seed development, such as PHERES1 (Kohler et al., 2003; Villar et al., 2009) and ADMETOS
449
(Kradolfer et al., 2013). Thus, there are new Brassicaceae-specific genes involved in seed
450
development that are regulated by the FIS-PRC2 complex. The functional innovation of the FIS-
451
complex appears to have rewired, to some extent, the regulatory pathway of seed development
452
specific to Brassicaceae.
453
Simultaneous gene duplication events, such as polyploidy, give rise to pairs of duplicated
454
genes that can then co-diverge (Shan et al., 2009). Many of the genes that are PRC2 targets,
455
included in the previous paragraph, were derived by the alpha whole genome duplication. FIS2,
456
MEA, and VEL2 also were derived from that WGD. Thus this study illustrates the potential of
457
concerted divergence after simultaneous gene duplication to affect functions as well as regulation
458
of other genes.
459
Materials and Methods
460
461
Comparing expression specificity and detecting co-expression using microarray data
462
analyses
463
Two sets of ATH1 microarray data from Arabidopsis thaliana were obtained: the Arabidopsis
464
development atlas (ADA) from the TAIR website (http://www.Arabidopsis.org/), which included
465
63 different organ types and developmental stages (Schmid et al., 2005), and the Arabidopsis
466
seed development atlas (ASA) from the Goldberg Lab Arabidopsis thaliana Gene Chip Database
467
(http://estdb.biology.ucla.edu/genechip/), which included 42 different tissue types from seed
468
developmental stages (Le et al., 2010). The data were GC-RMA normalized using the gcrma
469
package in R. We used the expression specificity (τ) defined by Yang and Gaut (2011) to
470
describe the expression patterns of FIS2, VRN2, MEA and SWN: τ =
471
where n is the total number of samples (63 or 42) and S(i,max) is the highest log2 transformed
472
expression values for gene i across the n organ types. High values of expression specificity
473
indicate genes with expression limited to few organ or tissue types or developmental stages,
474
while low values of expression specificity indicate broad expression of genes with similar
475
expression levels in most of the organ or tissue types and developmental stages. To test if there is
476
any significant difference of expression specificity between any two of the four genes, we
[
( , )/ ( ,
)]
,
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477
applied 1000 Monte Carlo randomization tests to each two-gene comparison. For the Monte
478
Carlo randomization test, we computed the following statistic: DIF = |τGENE1-τGENE2|, where DIF
479
indicates the absolute difference of expression specificity between two genes. Then, we
480
compared the observed value (DIFobs) against the null distribution of simulated DIF value (=
481
DIFsim) from 1000 randomized data. If the null hypothesis is rejected, the expression specificity
482
of any two compared genes is significantly different. The cutoff of the significant P value was
483
set to 0.05.
484
In addition to the comparison of expression specificity among gene pairs, we applied the
485
Pearson correlation analysis to determine if the expression profile between any two genes
486
showed any evidence of co-expression (i.e. correlated expression across different organ types or
487
tissue types). Co-expression is determined when the Pearson correlation coefficient (r) is
488
significantly positive, and vice versa.
489
490
Inferring the ancestral expression states using RT-PCR
491
Total RNA samples of Arabidopsis thaliana, Tarenaya hassleriana (formerly known as Cleome
492
spinosa), Carica papaya, and Vitis vinifera were extracted from liquid N2 frozen tissue of five
493
organ types: root, stem, leaf (rosette leaves in Arabidopsis thaliana), flower, and seed (whole
494
siliques in Arabidopsis thaliana and Tarenaya hassleriana). A modified CTAB method was used
495
for RNA extraction (Zhou et al., 2011). The quality of each RNA sample was checked on 2%
496
agarose gels by electrophoresis, and the amount of each RNA sample was determined by a
497
Nanodrop spectrophotometer. After DNaseI (Invitrogen) treatment to remove residual DNA, M-
498
MLV reverse transcriptase (Invitrogen) was applied to the RNA samples to generate cDNA,
499
according to the manufacturer’s instructions. PCR was performed with cDNA templates to detect
500
the organ-specific expression of Arabidopsis FIS2/VRN2 and MEA/SWN paralogous pairs, as
501
well as orthologous genes in outgroup species for inference of the ancestral, pre-duplication,
502
expression states. Gene-specific primers were designed to amplify 250-1000 bp of the cDNA of
503
targeted genes (Table S2). For PCR reactions, the cycling programs were: preheating at 94℃ for
504
3 minutes; 30-35 cycles of denaturing at 94℃ for 30 seconds, annealing at 53-56℃ for 30
505
seconds, elongation at 72℃ for 30 seconds or 1 minute, and a final elongation at 72℃ for 7
506
minutes. PCR products were checked on 1% agarose gels, and sequenced to confirm identity.
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507
508
Identifying epigenetic marks associated with the studied genes
509
We investigated the epigenetic modifications around the genomic regions of Arabidopsis FIS2,
510
VRN2, MEA and SWN. We also used EMF2 and CLF, which are members of the FIS2/VRN2 and
511
MEA/SWN families, respectively, to help assess the ancestral state. For DNA methylation, we
512
obtained data from Schmitz et al. (2013), Stroud et al. (2013) and Zemach et al. (2013) from
513
CoGe (https://genomevolution.org/CoGe/), visualized by JBrowse in Araport
514
(https://www.araport.org/). Analyzed data included assayed genomic DNAs from leaves in
515
Schmitz et al. (2013) and Stroud et al. (2013), and assayed genomic DNAs from seedlings and
516
roots in Zemach et al. (2013), which were all vegetative organs. Cytosine methylation at CpG
517
sites was analyzed along the genomic region of a target gene. For histone methylation, we
518
extracted tiling-array data from seedlings from Roudier et al. (2011) and ChIP-on-chip data from
519
wild type and fie mutant seedlings from Bouyer et al. (2011). Four histone marks were analyzed:
520
tri-methylation of lysine 27 on histone H3 (H3K27me3), tri-methylation of lysine 4 on histone
521
H3 (H3K4me3), di-methylation of lysine 4 on histone H3 (H3K4me2) and tri-methylation of
522
lysine 36 on histone H3 (H3K36me3). The epigenetic features in Arabidopsis seedlings were
523
compared among the paralogous genes in a family and between the two interacting gene families.
524
525
Detecting accelerated sequence evolution and positive selection by Ka/Ks analyses
526
To analyse the selection acting on the gene pairs FIS2/VRN2 and MEA/SWN, several rate
527
analyses were performed using Codeml in the PAML package (Yang, 2007). We obtained the
528
sequences of the four genes from Arabidopsis thaliana, as well as some other Brassicaceae
529
species, including Arabidopsis lyrata, Arabidopsis halleri, Capsella rubella, Brassica rapa,
530
Brassica oleracea, Eutrema salsugineum (formerly known as Thellungiella halophila), and
531
Schrenkiella parvula (formerly known as Thellungiella parvula). We also identified orthologous
532
sequences, by reciprocal best BLAST hits, from species outside of the Brassicaceae including
533
Tarenaya hassleriana (formerly known as Cleome spinosa), Carica papaya, Gossypium
534
raimondii, Theobroma cacao, Citrus sinensis, Populus trichocarpa, Ricinus communis, and
535
Manihot esculenta, Vitis vinifera from PLAZA v3.0 Dicots
536
(http://bioinformatics.psb.ugent.be/plaza/versions/plaza_v3_dicots/; Proost et al., 2015),
23
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537
Phytozome v10 (http://phytozome.jgi.doe.gov/pz/portal.html; Goodstein et al., 2012), BRAD
538
database (http://brassicadb.org/brad/; Cheng et al., 2011) and NCBI’s GenBank. Gene orthology
539
was later confirmed by comparing the topology of the gene phylogeny to the species tree.
540
Alignments of amino acid sequences were generated using MUSCLE under default parameters
541
(Edgar, 2004), and then reverse translated into codon alignments using the customized Perl script.
542
We generated the alignments for the full length of the two gene families, as well as some
543
documented functional domains, including the VEF and C2H2 domains in the FIS2 and VRN2
544
genes, and the C5, SET, SANT and CXC domains in the MEA and SWN genes. Phylogenies of
545
the two gene families were analyzed by RAxML v.7.0.3 with GTR as the substitution matrix
546
(Stamatakis, 2006). ML trees of the two gene families were generated based on codon
547
alignments.
548
We first used a phylogeny-based free-ratio test to estimate branch-wise Ka/Ks ratios
549
along the phylogenetic tree branches. For the full-length FIS2/VRN2 genes we implemented four
550
different models to test if the Ka/Ks ratios of the Brassicaceae FIS2 clade and the Brassicaceae
551
VRN2 clade display an asymmetric pattern, and how conserved they are compared to the
552
orthologous genes. The first model (Model I: one-ratio model) assumes that all the genes have
553
the same Ka/Ks ratio, bearing the hypothesis that all genes are under the same level of selection.
554
The second model (Model II: two-ratio model-1) assumes that the Brassicaceae VRN2 clade and
555
the orthologous genes have the same Ka/Ks ratio, but the Brassicaceae FIS2 clade can have a
556
different one, suggesting that the Brassicaceae VRN2 clade reflects the ancestral selection but
557
FIS2 evolved in a different manner. The third model (Model III: two-ratio model-2) assumes the
558
duplicated FIS2 and VRN2 clades in Brassicaceae have the same Ka/Ks ratio, while the orthologs
559
can have a different ratio, which is a hypothesis that the two Brassicaceae copies evolved at the
560
same rate. The fourth model (Model IV: three-ratio model) assumes that the two Brassicaceae
561
branches have different Ka/Ks ratios, and thus the two genes evolved at different rates, with the
562
third Ka/Ks ratio for the orthologous branches. A set of likelihood ratio tests were applied, where
563
twice the different of likelihood values was calculated and compared against a chi-square
564
distribution with the degree of freedom (df) set at one: comparison between Model II and Model
565
IV can tell if the selection on the Brassicaceae VRN2 is significantly different from the
566
orthologous genes; and comparisons between Model I and Model II, as well as between Model
567
III and Model IV is to see if the selection on the Brassicaceae FIS2 is different from the
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568
Brassicaceae VRN2 and/or the orthologous genes. When Model II fits better than Model I, and
569
Model IV fits better than Model III with statistical support, the evolutionary rate of the
570
duplicated pair in Brassicaceae is considered to evolve asymmetrically. The same analyses were
571
performed on the functional domains of the FIS2/VRN2 genes, and the full-length MEA/SWN
572
genes and their functional domains (Table S1). We also applied a branch-site model to detect
573
positively selected sites along FIS2 as well as MEA. Test 2 of ModelA with the Bayes Empirical
574
Bayes analysis was applied to identify amino acid sites with a high posterior probability of
575
positive selection (Zhang et al. 2005).
576
577
578
579
580
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581
Table(s)
582
Table 1. Histone methylation of studied genes.
583
H3K27me3
VEF
genes
SET
genes
VEL
genes
FIS2
VRN2
EMF2
x
MEA
SWN
CLF
x
VEL2
VEL1
x
H3K4me3
H3K4me2
H3K36me3
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
584
585
x’s indicate presence of a particular type of histone methylation
586
587
588
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589
SUPPLEMENTAL DATA
590
Fig. S1. Permutation test for microarray data to detect the difference in expression profile for all sets of
591
comparisons of gene pairs in the ADA and ASA datasets.
592
Fig. S2. Structures of FIS2 and VRN2, along with MEA and SWN in Brassicaceae and other eurosids.
593
Fig. S3. Ka/Ks ratios of full-length FIS2/VRN2 and MEA/SWN genes and functional domains.
594
Fig. S4. Positive selection on specific sites of MEA and FIS2 genes.
595
Table S1. Ka/Ks ratios under different branch models for full-length FIS2/VRN2 and MEA/SWN genes
596
and functional domains.
597
Table S2. Gene-specific primers used in this study.
598
599
600
Figure Legends
601
Figure 1. Two PRC2 complexes in Brassicaceae, the VRN-complex and the Brassicaceae-
602
specific FIS-complex, arose by the alpha whole genome duplication where VRN2 duplicated to
603
form FIS2, and SWN duplicated to form MEA.
604
605
Figure 2. A. Organ/tissue-specific expression indices based on two sets of microarray data. A
606
large value indicates expression is restricted to fewer organ or tissue types while a low value
607
indicates broad expression. B. Correlation of expression profile of each gene pair. Left: ADA set
608
(63 organ types and developmental stages); right: ASA set (42 seed tissue types and
609
developmental stages). Black arrows indicate a positive correlation and grey arrows indicate a
610
negative correlation. The thickness of arrows indicates the level of the correlation coefficient.
611
The correlation coefficient and p-value of expression profile of each gene pair are labeled along
612
the arrows. Bold values indicate positive correlation.
613
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614
Figure 3. RT-PCR assays indicate that FIS2 and MEA have lost the ancestral vegetative
615
expression pattern after duplication. Plus signs indicate reactions with reverse transcriptase and
616
minus signs indicate controls with no reverse transcriptase. Species abbreviations include: At -
617
Arabidopsis thaliana, Th - Tarenaya hassleriana, Cp - Carica papaya, and Vv - Vitis vinifera.
618
619
Figure 4. DNA methylation at the genomic region of the VEF-domain genes and SET-domain
620
genes. CLF and EMF2 are ancient paralogs of SWN and VRN2, respectively. For each gene, four
621
rows represent four replicates, and the dashed line separates 1500 bp upstream of the
622
transcription start site. Vertical bars in each row represent the level of methylation.
623
624
Figure 5. Ka/Ks values of the interacting domains: VEF domain in FIS2/VRN2 and C5 domain
625
in MEA/SWN. Estimated average Ka/Ks ratio of each clade is shown between the two trees. The
626
values above branches are Ka/Ks ratios (where no value suggested the lack of power to detect the
627
accurate Ka/Ks ratio in the PAML analysis). The black dots indicate the alpha WGD at the base
628
of the Brassicaceae. The scale bars indicate 0.1 substitution per codon. Species abbreviations
629
include: At - Arabidopsis thaliana, Al - Arabidopsis lyrata, Cr - Capsella rubella, Sp -
630
Schrenkiella parvula, Es - Eutrema salsugineum, Br - Brassica rapa, Bo - Brassica oleracea, Th
631
- Tarenaya hassleriana, Cp - Carica papaya, Gr – Gossypium raimondii, Tc - Theobroma cacao,
632
Pt - Populus trichocarpa, Rc - Ricinus communis, Me - Manihot esculenta and Vv - Vitis
633
vinifera.
634
635
Figure 6. VEL2 and VEL1 expression and sequence evolution. A. Organ/tissue specificity of
636
VEL genes. B. Correlation of expression profile between VEL genes and PRC2 core components.
637
Left: ADA set (63 organ types and developmental stages); right: ASA set (42 seed tissue types
638
and developmental stages). Black arrows indicate positive correlation, and grey arrows indicate
639
negative correlation. The thickness of arrows indicates the level of the correlation coefficient.
640
The correlation coefficient and p-value of expression profile of each gene pair are labeled along
641
the arrows. Bold values indicate positive correlations. C. DNA methylation at the genomic
28
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642
region of VEL genes (as in Fig.4). D. Ka/Ks values of the VEL genes. Average Ka/Ks ratio of
643
each clade is shown. The black dot at the node indicates gene duplication events.
644
645
Figure 7. Schematic diagrams illustrating models of protein complex divergence. Colors indicate
646
conservation vs. divergence (could be neofunctionalization, subfunctionalization, loss of partial
647
function, and other types of divergence). A. Single-gene-duplication and divergence: a single
648
gene (dark blue) in a complex is duplicated. After duplication there is subsequent divergence
649
(light blue vs. red) of the ancestral gene (dark blue) to give rise to divergent protein complexes.
650
B. Simultaneous-gene-duplication and concerted divergence: two (or more) genes (dark green +
651
dark blue) were duplicated simultaneously. After duplication there is parallel divergence (light
652
green + light blue vs. yellow + red) to give rise to divergent protein complexes. C. The PRC2
653
complexes in this study are an example of simultaneous-gene-duplication and concerted
654
divergence.
655
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